Art of butyl-acetonic fermentation



Patented May 23, 1933 UNITED STATES PATENT OFFICE DAVID A. LEG-G, OITERRE HAU'IE, INDIANA, ASSIGNOR T0 COMMERCIAL SOLVENTS CORPORATION, OFTEBRE HAU'IE, INDIANA, A CORPORATION: OF MLABYLAND ART OF BUTYL-ACETONICFERMENTATION No Drawing.

My invention relates to the process of preparing butyl alcohol andacetone b fermentation. More particularly, it re ers to a method ofobtaining bacterial cultures capa- 5 ble of'producing these substances,which are not subject to periods of sluggish fermentation. i

Weizmann in United States Patent No.

1,315,585 of September 9, 1919, has described 1) a process wherebynormal butyl alcohol, acetone, and ethyl alcohol in approximately theproportions of 6:3: 1 are obtained by fermenting a carbohydrate mashwith an organism which has been variously classified under the names ofB. amylobacter, B. granulobacter pectino'vomm, Glostfidiu'm actobutyZi-.cum (Weizmann) ,etc. On inoculating a sterile carbohydrate, as forexample, corn meal mash with a culture of this organism and allowing itto ferment at about 37 C. certain typical reactions usually ensue if theculture is a normal healthy one and no contaminating organisms capableof reducing excessive acid production are present in appreciablequantities. As a general rule, such a normal fermentation is completedin the course of from 24 to 60 hours, during which time a slimy firmhead or cap of unfermented material is formed and the underlying liquorbecomes somewhat cloudy. During thecourse of the fermentation theacidity of the medium rises v to a peak requirin for 10 c. c. ofmashbetween' 4 and 6 c. c. of /10 soda solution to neutralizeto'phenolphthalein and then breaks from the peak within a few hours toa. final acidity of 2 to 3 c. c. of 0.1N sodium hydroxide per 10 c. c.

In the commercial scale production of butyl alcohol and acetone byfermentation 40 two outstanding types of fermentation difliculties havebeen encountered. One of these has been for convenience designated ascontaminated fermentations andiscausedbythe accidental presence of otherforms of bacteria than those producing butyl alcohol and acetone. Themost common forms of contaminating organisms met with in this type offermentation are the so-called thermobacterium group of lactic acidorganisms including.

B. columns, oasez', etc., the group of organfermentation to becompleted. S u

Application filed November 27, 1928. Serial No. 410,258.

isms producing mannitol and ethyl alochol.

The presence of such organisms causes either I or both of two thingsequally detrimental to which may inhibit the growth and develop ment ofthe butyl organisms. In such a case the time required for thefermentation to be completed is considerably lengthened and at the sametime poor yields are obtained due to the action of the stunted orweakened culture. "In addition to the harmful effect of theseforeignorganisms upon the butyl organlsm itself, their presence leads toanother equally important undesirable efiect. Bemg present in such largequantities they consume a large proportion of the carbohydrate designedfor the use of the butyl organisms, with the result that the finalyields of butyl alcohol and acetone from a particular lot ofcontaminated mash are materially reduced below those obtained from anormal uncontaminated culture. Theuse of contaminated culture alsopresents certain other disadvantagei which need not be mentioned at thispom In the past, poor fermentations and yields have at times beenobtained even when no signs of contaminating organisms could bedetected. Marked differences are observed between normal fermentationsand these as they sometimes are termed sluggish fermentations. In thecase of the latter, large fermenters containing 40-50 thousand gallonsof an 8%9% corn meal mash require -100 hours and sometimeslon er for theish fermentations show a marked inhi ition or often complete absence ofhead formation in from 24 to 48 hours, the liquor usually being clearand mobile instead of cloudy and vis= cous as in a normal fermentation.The acidity of the fermenting medium rises to a peak of between 5 and 7c. c. of 0.1N sodium h droxide per 10 c. c. of mash (phenolphthalei nand generally the final yields of butyl alcohol and acetone are from 10%to 15% below those obtained from normal fermentations. Even tho nomicroscopic evidence of the presence of other forms of living organismsis available, the recurrent and epidemic character of the sluggis'hnesssuggests that it is a disease of the butyl organisms caused by thepresence of an ultra-microscopic organism possibly of parasiticcharacter.

In United States Patent No. 1,668,814 granted May 8, 1928, I havedisclosed a method' whereby the difficulties caused by sluggishfermentation of the above-described character may be successfullyovercome. This method consists substantially oi immunizing the butylorganisms by repeatedly subcultivating them in the presence of thefiltrate from a sluggish fermentation, the cultures being heat shockedbefore each transfer. In carrying out the process in this manner, asterile carbohydrate mash consisting for example of 6% corn mash isprepared and inoculated with a spore culture of butyl-acetonic bacilli.It is then heat-shocked for three minutes at 100 C. after which there isadded a small portion (one or more drops) of clear liquid obtained froma carbohydrate mash undergoing a sluggish butyl-acetonic fermentation byfiltering the mash first to remove all coarse suspended solids andsecond thru a Berkefeld or other bacterial filter to remove bacteria.Fermentation is then allowed to proceed at a temperature of about 37 C.for four or more days, i. e., until spores have developed. This sporeculture is then used to inoculate fresh corn mash which is againheat-shocked for about three minutes at 100 G, and treated with twodrops of the filtrate as previously mentioned. lhe second fermentationis allowed to proceed as before and additional transfers with shockingand with filtrate addition are made.

At the end of from about 10 to about 17 such instead of inoculating asterile carbohydrate o. a culture taken directly from a Eerme lg mash."heat-shocking, adding a few drops of liquid containing the supposedultra-virus, allowing fermentation to take place, etc., I have now foundthat 1 may proceed as follows. A sterile carbohydrate mash consisting,for example, of 6% corn mesh is prepared and inoculated with a pureculture of butyl-acetone bacilli." To this is then added a smallportion, say one or more drops, of clear liquid obtained from acarbohydrate mash undergoing .a sluggish butyl-acetonic fermentation byfiltering the mash first to remove all solids and second thru aBerkefeld or other bacterial filter to remove bacteria. Fermentation isthen allowed to proceed at a temperature of about 37 C. When thefermentation is complete the mash is allowed to stand for a period offrom 4 days to a month for the purpose of allowing all vegetativeorganisms present to die, thus leaving in the mash only the spores ofthe butyl-acetonic bacilli which remained sufliciently vigorous afterbeing attacked by the ultra-virus organism to reach the spore-formingstage. The general assumption is that the weaker and more susceptibleorganisms will have been so attacked and further weakened by the actionof the ultra-viruswhich, it will be recalled, is thought to be ofparasitic character living on the butyl-acetonic organism and not uponthe carbohydrate-that only those organisms which are least susceptibleto the action of the ultra-virus organisms will reach the spore-formingstage of development.

The presence of vegetative forms of organisms remaining in the mash maybe determined by using a small amount of the mash as the inoculum for asterile 6% corn mash. If fermentation commences in less than 1246 hoursvegetative cells are probably still'alive, and it is necessary to allowthe mash to stand. for a further period oftime. I When it is determinedthat all of the vegetative forms are dead, the operation may be repeatedsubstantially as has just been described, using the butyl-acetonicbacilli spores obtained from the previous fermentation as the inoculumfor a new lot of sterile 6% corn mash and again adding a small amount ofliquid containing the ultra-virus organism, allowing fermentation totake place and the resulting mash to stand until all vegetative cellsare dead. The operation may then be repeated a sutlicient number oftimes to render the resulting butyl-acetonic bacilli completelyresistant to the action of the ultra-virus organisms, and comparativetests of treated and untreated cultures show that sluggish fermentationand/ or lowered yields are not produced by the addition of theultra-virus organisms to the treated culture. Ordinarily from about 10to about l 'l'such treatments are sufficient to give a completelyimmunized culture. In some cases, however, it has been found that adilierent number of treatments is required, and it is specificallyunderstood that I do not restrict myself to the use of any particularnumber of sub-cultivations.

Similar results may be obtained in a considerably shorter length of timeby adding to a culture, prepared as above described, after it hasattained 48 to 72 hours growth, an amount of a toxic material sufiicientto destroy all vegetative forms of organisms present but not sufficientto have any ap reciable effect upon the spore forms. Suita le materialsare mineral acids, phenol, cresols, mercurochrome, or other substancehaving a bactericidal action. The amount of the toxic material requiredwill depend upon the particular material used, the time of treatment,etc. Whatever material is em loyed, care should be taken not to usesufli dient of the toxic material so that at the dilution attained inthe succeeding transfer it will still have an appreciable toxic effectupon the organisin.

As examples of satisfactory methods of employing toxic substances forthe purposes of destroying the vegetative forms of organism in my newmethod of immunizing butyl-acetonic bacilli, I may add to the culturebeing treated with the ultra-virus organism and which has been allowedto ferment for 48 to 72 hours, say 0.1% of sulphur dioxide as sodiumsulfite, or 0.5% or more of phenol or cresol. After allowing thesematerials to act for six hours or more a portion of the resulting mashis employed to inoculate a new lot of sterile 6% corn mash to which isthen added a few containing the ultra-virus organism. ermentation isthen allowed to take place as usual and the operation repeated, ashitherto.

specified, a suflicient number of times to rencharacter specifie der thebutyl-acetonic bacilli completely resistant against the ultra-virusorganism.

Instead of usin a toxic material of the d above, sufiicient mineral acidis added to the mash which has been allowed to ferment for 48-72 hours,to lower its hydrogen ion concentration to a value of about pH2.5. Themash is then allowed to stand for two hours or longer and a portion ofthe resulting mash used as the inoculum for a new lot of sterile mash.

Still another method of destroying the vegetative organisms is to exposethe clear liquid from a culture being immunized to the action ofultra-violet light. For example, if the clear liquid is exposed in a 1centimeter quartz tube to the action of the Uviarc mercury lam at adistance of 10 inches for a period 0 will be destroyed and only sporesremain.

Shaking in a shaking machine and drying out at low temperatures areother alternative methods which may also be employed in my process fordestroying the vegetative cells and leaving only the spores.

Cultures of the butyl-acetonc bacilli, which have been immunized or maderesistant against the agency causing sluggish fermen- 'tation by any of.the different modifications of my new process described above havedistinct advantages over other similar cultures drops of the lig uid 10minutes the vegetative forms which have not been subjected to theimmunizing treatment, in that they give much more constant. and uniformresults than is possible from non-immunized cultures. In

general higher yields of'solvents are prouced by these immunizedcultures both under ordinary conditions and during periods whennon-heated cultures show symptoms of sluggish fermentation and give low.ther a greater or lesser number of times than specified, in order to berendered satisfactorily immunized. In place of sterile corn mash I mayemploy any fermentable carbohydrate of either saccharin oramylaceouslcharacter.

Now having described my invention, I

.claim the following as new and novel.

1. Process for producing an immunized culture of but l-acetone bacilliwhich comprises repeate ly sub-cultivating said bacilli in carbohydratemedia in bohydrate which is undergoing sluggish butyl-acetonicfermentation, and eliminating the vegetative cells at ordinarytemperatures previous to each sub-cultivation.

2. Process for producing an immunized culture of butyl-acetone bacilliwhich-com: prises repeatedly sub-cultivating said bacilli incarbohydrate media in the presence of a Berkefeld filtrate of acarbohydrate mash which is undergoing sluggish butyl-acetonicfermentation, and eliminating the vegetative cells at ordinarytemperatures previous to each transfer.

3. Process for producing an immunized culture of butyl-acetone bacilliwhich comprises sub-cultivatingrsaid bacilii ten or more times in acarbohydrate medium in the presence of a portion of a Berlxefeldfiltrate of a carbohydrate mash which s undergoing sluggish butl-acetonie fermentat1on, and eliminating t e vegetative cells atordinary temperatures previous to each sub-cultivation.'

4. In a process for producing an immunized culture of butyl-acetonebacilli, the step which comprises eliminating the vegetative cells atordinary temperatures previous to each sub-cultivation.

the presence of carbohydrate which is undergoing sluggish butyl-acetonicfermentation, and eliminating the vegetative cells previous to eachsub-cultivation at temperatures not substantially above normalfermentation temperature.

6. Process for producing an immunized culture of butyl-acetone bacilliwhich comprises repeatedly sub-cultivating said bacilli in carbohydratemedia in the presence of a Berkefeld filtrate of a carbohydrate mashwhich is undergoing sluggish butyl-acetonic fermentation, andeliminating the vegetative cells previous to each sub-cultivation attemperatures not substantially above normal termentation temperature.

7. Process for producing an immunized culture of butyl-acetone bacilliwhich comprises sub-cultivating said bacilli ten or more times in acarbohydrate medium in the presence of a portion of a Berket'eldfiltrate of a carbohydrate mash which is undergoing sluggishbutyl-acetonic fermentation, and eliminating the vegetative cellsprevious to each sub-cultivation at temperatures not substantially abovenormal fermentation temperature.

8. In a process for producing an immunized culture of butyl-acetonebacilli, the step which comprises eliminating vegetative cells previousto each sub-cultivation at temperatures not substantially above normalfermentation temperature.

9. Process for producing an immunized culture of butyl-acetone bacilliwhich comprises repeatedly sub-cultivating said bacilli in carbohydratemedia in the'presence of carbohydrate which is undergoing sluggishbutyl-acetonic fermentation, and eliminating the vegetative cellsprevious to each sub-cultivation by adding to the culture a quantity ofa toxic agent sutlicient destroy the vegetative cells, but insufiicientto destroy the spores.

10. Process for producing an immunized culture of butyl-acetone bacilliWl'lIlCll comprises repeatedly sub-cultivating said bacilli incarbohydrate media in the presence of a Berkefeld filtrate of acarbohydrate mash which is undergoing sluggish butyl-acetonicfermentation, and eliminating the vegetative cells previous to eachsub-cultivation by acid ing to the culture a quantity of a tonic agentsuih'cient to destroy the vegetative cells, but

J insufiicient to destroy the spores.

l1. -iProccss for producing an immunized culture of butyl-acetonebacilli which comprises sub-cultivating said bacilli ten or amoretimesin a carbohydrate medium in the presence or a portion oi a Berheieldtil-traits of a carbohydrate mash which is undergr sluggishbutyl-acetonic fermentation,

eliminating the vegetative cells previous to each sub-cultivation byadding to the culture a quantity of a toxic agent suiiicient to de stroythe vegetative cells, but insuflicient to destroy the spores.

12. In a process for producin an immunized culture of butyl-acetoneacilli, the step which comprises e'iminating vegetative cells previousto each sub-cultivation by adding to the culture a quantity of a toxicagent sufficient to destroy the vegetative cells, but insufiicient todestroy the spores.

13. Process for the production of butyl alcohol and acetone whichcomprises preparing a sterile carbohydrate mesh and inoculating saidmash with a culture of butyl-acetone bacilli, the said culture havingbeen previously sub-cultivated in carbohydrate media in the presence ofcarbohydrate which is undergoing sluggish butyl-acetonic fermentation,the attenuated vegetative cells having I been eliminated previous toeach transfer in said sub-cultivating process at temperatures notsubstantially above normal fermentation temperature.

14:. Process for the production of butyl alcohol and acetone whichcomprises preparing a sterile amylaceous mash and inoculating said mashwith a culture of butyi-acetone bacilli, the said culture having beenpreviously sub-cultivated in carbohydrate media in the presence ofcarbohydrate which is undergoing sluggish butyl-acetonic fermentation,the vegetative cells having been eliminated previous to eachsub-cultivation in said sub-cultivating process at temperatures notsubstantially above normal fermentation temperature.

In testimony whereof I affix my signature.

DAVID A. LEGG.

CERTIFICATE OF CORRECTION.

Patent No. 1,911,174. May 23, 1933.

the word "attenuated", and line 10, for "transfer" read"sub-cultivation"; and

that the said Letters Patent should be read with these correctionstherein-that the same may conform to the record of the casein the PatentOffice. 7

Signed and sealed this 15th day of August, A. D. 1933.

M. J. Moore (Seal) Acting Commissioner of Patents.

